In humans, exposure to ionizing radiation occurs through natural sources (such as ultraviolet or other electromagnetic radiation or cosmic radiation from the sun, stars or terrestrial radioactive sources in the earth's crust) or from various man-made sources. The primary exposure from man-made sources comes from diagnostic x-rays and radionuclide studies, dental x-rays, and therapeutic techniques (such as anticancer radiotherapy) or to a lesser extent due to fallout from atmospheric atomic weapons testing, nuclear power plants and through occupational exposure. Ionizing radiation has an adverse effect on cells and tissues, primarily through cytotoxic effects. A major way in which most forms of ionizing radiation damages biomolecules and cells is through a process called indirect action involving an interaction with water to produce toxic active oxygen species (OH—, —O2−, or H2O2). A second mechanism, called direct action, involves direct effects on DNA.
Diminution of the deleterious effects of ionizing radiation by chemoprotection would be important to a number of diverse groups, including the general population exposed to cosmic and terrestrial irradiation, to patients given diagnostic and dental x-rays, to workers exposed to irradiation, to groups exposed to radiation through accidents or acts of terrorism, to astronauts and airline personnel exposed to “extra” cosmic irradiation, and to patients given radiation to treat cancer. Approximately 60% of all cancer patients receive radiation as part of their therapy, and the harm to the normal tissues often limits the radiation dose which can be administered to the tumor. With regard to ultraviolet radiation, the increasing prevalence of melanomas, pterygium and cataracts suggest the necessity of prophylactic measures to reduce these problems.
Evidence is accumulating that the etiology of many degenerative diseases that afflict humanity includes free radical reactions. Examples of such degenerative diseases include atherosclerosis, cancer, inflammatory joint disease, arthritis, autoimmune diseases, asthma, diabetes, senile dementia, Alzheimer's disease, Parkinson's disease, multiple sclerosis, muscular dystrophy, ischemia, stroke, congestive heart failure and degenerative eye diseases. The process of biological ageing might also have a free radical element. Most free radical damage to cells involves oxygen free radicals or, more generally, activated oxygen species, which include non-radical species such as singlet oxygen and hydrogen peroxide as well as free radicals.
The eye is one organ with intense activated oxygen species activity, and it requires high levels of antioxidants to protect its unsaturated fatty acids. Glaucoma is an example of a degenerative disease of the eye that can lead to retinal cell death. Glaucoma is a widespread ocular disease of unknown etiology that can lead to eventual blindness due to gradual loss of retinal ganglionic cells (RGC). The majority of glaucoma patients have elevated intraocular pressure (IOP), and drugs capable of lowering IOP, including beta adrenergic blockers, prostanoids, cannabinoids and carbonic anhydrase inhibitors, are used clinically to reduce the impact of the disease. A significant percentage of glaucoma patients, however, do not demonstrate elevated IOP, but still show gradual reduction in ocular function due to retinal cell death. Recently, attention has focused on attempts to use neuroprotective agents to increase the survival of the retinal cells, a strategy that should be effective in all glaucoma patients.
It is now evident that ganglion cells are dependent on a variety of eurotrophins, but primarily on brain-derived neurotrophic factor (BDNF). An adult ganglion cell takes up secreted BDNF from its respective target neuron and transports it along its axon back to the cell body in the retina. Glaucoma is now thought to block this retrograde flow of BDNF by blocking axoplasmic transport at the site of the lamina cribrosa (Nickells R W., J. Glaucoma 1996; 5:345-356). It is not precisely known how long a ganglion cell can survive without its BDNF supply, but tests conducted in culture suggest that it is only a matter of days. One obvious neuroprotective strategy that has been considered for glaucoma treatment is to provide a different source of BDNF for the ganglion cells. Another damaging stimulus associated with glaucoma is the release of excitotoxins. These molecules are actually excitatory amino acids, such as glutamate, that are normally used by neurons as neurotransmitters. At high local concentrations, however, these normally benign molecules activate a highly toxic response in nearby cells (hence the derivation of the word “excitotoxin”). Like neurotrophins, excitotoxins interact with receptors on the cell surface. There are three known sub-types of glutamate receptors found on neurons, but the one that appears to play the biggest role in the excitotoxic effect is the N-methyl-D-aspartate (NMDA) receptor. Elevated levels of glutamate have been detected in the vitreous of both human glaucoma patients and monkeys with experimental glaucoma (Dreyer E B, Zurakowski D, Schumer R A, Podos S M, Lipton S A., Arch. Opthalmol. 1996; 114:299-305).
Transition metals such as iron and copper are known to generate cytotoxic free radicals, whereas iron-regulating proteins such as transferrin, ceruloplasmin, and ferritin have been shown to act as anti-oxidants, counteracting the toxic effects of these metals. Iron and copper cations are released from tissues during ischemia and in association with a variety of disease processes. Tissues deprived of blood and oxygen undergo ischemic necrosis or infarction with possible irreversible organ damage. Even if the flow of blood and oxygen is restored to the organ or tissue (reperfusion), the organ does not immediately return to the normal pre-ischemic state. Post-ischemic dysfunction may be due to the generation of oxygen free radicals in the stunned organ. The re-entry of neutrophils during reperfusion can create free radical damage due to the hyper-reactivity of leukocytes. Iron and copper cations are known to catalyze hydroxy free radical formation. The chelator ethylenediaminetetraacetic acid (EDTA) is known to reduce lipid peroxidation from ionizing radiation. (Ayene-S I; Srivastava-P N Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 1985 August; 48(2): 197-205). Providing anti-oxidants and reducing equivalents to ischemically stressed tissue allows recovery of the mitochondria, which is crucial in restoring normal calcium homeostasis and tissue function.
It is further known that metal cations, such as Ca++, that are not first transition series elements perform important functions in the body, but may also be involved in a number of pathogenic processes, such as calcium overload of the heart that occurs during ischemia and reperfusion. Such metal cations can compete with cations of first transition series elements for chelation by a chelator that has a high affinity for Ca++, and thereby interfere with the chelation of first transition series cations by the chelator. Moreover, chelation of cations such as Ca++ can impair the normal functions of these cations in the body. Therefore, the use of chelators must be tempered with the knowledge of the role of various cations in the disease process to be treated and the selection of specific chelator affinities to remove the offending cations without creating additional ionic imbalances.
Numerous attempts have been made to reduce normal tissue damage while still delivering effective therapeutic doses of ionizing radiation to the cancerous tissue. These techniques include brachytherapy, fractionated and hyperfractionated dosing, complicated dose scheduling and delivery systems, and high voltage therapy with a linear accelerator. However, such techniques can, at best, only attempt to strike a balance between the beneficial effects of killing the cancer cells and undesirable effects of the radiation to the normal tissues. There is much room for improvement in the therapeutic ratio, which is the ratio of a measure of damage to the tumor divided by the damage to the normal tissues.
Attempts to mitigate the catalytic effectiveness of iron and copper cations by administering iron chelating siderophores such as deferoxamine to form complexes with these cations have not been unequivocally successful in inhibiting tissue damage from hydroxy free radicals in vivo. Siderophores such as deferoxamine are poor chelators for copper cations. Although present in the body in much lower concentrations than iron, copper is far more active than iron in catalyzing hydroxyl radical formation.
Polyethylene glycol (PEG) and polyethylene glycol monomethyl ether (MP) have also been found to reduce tissue injury, although the mechanism is obscure. PEG is an amphiphilic polymer H(OCH2 CH2)nOH that consists of a mixture of homologs with a range of similar molecular weights. Thus, for example, MP 350 possesses an average molecular weight 350, and consists of a mixture of homologs with n=4 to 9, and a median n=7. The lower molecular weight polymers PEG 200-600 are absorbed through the gastrointestinal tract when ingested orally and excreted unchanged in the urine. PEG is absorbed along with water directly through the intestinal mucosal cell membrane.
PEG 200-600, being nontoxic and biologically inert, has often been used as a vehicle for administration of drugs insoluble in water. In several investigations, the PEG vehicle alone was empirically found to exhibit significant biological activity, leading to further studies of low molecular weight PEG. For example, PEG 400, when given intraperitoneally (IP) either before or shortly after x-irradiation of mice, conferred significant protection against lethality and morbidity (Shaeffer and Schellenberg, Int. J. Radiat. Oncol. Biol. Phys., 10:2329, 1984; Shaeffer, et al., Radiat. Res., 107:125, 1986). PEG 300 IP was shown to reduce the CNS sequelae of experimental concussive brain injury (Clifton, et al., J. Neurotrauma, 6:71, 1989).
PEG with a molecular weight around 400 is thus a uniquely nontoxic substance that exhibits a protective effect against injury to tissues. However, PEG with a molecular weight greater than 700 is poorly absorbed through the GI tract. The mechanism of the protective action of lower molecular weight PEG has not been established, but probably involves interaction of PEG with the surface of lipid membranes or protein components. PEG aggregates near cell membranes, reduces water polarity at membrane surfaces, and increases hydrophobic interactions (Hoekstra, et al., J. Biol. Chem., 264:6786, 1989).
It is known that certain MP chelates can be effective iron chelators. For example, it is known that MP can be linked with iminodiacetate terminus (MIDA). Other chelates modified with MP include MP 550-deferoxamine (ferrioxamine), prepared by reacting MP molecular weight 550 with carbonyldiimidazole, followed by reaction of the resulting imidazolecarbonyl ester with deferoxamine base, forming a urethane linkage. The material was produced as a chelate for gadolinium, to be used as a renal magnetic resonance contrast agent (Duewell, et al., Invest. Radiol., 26:50, 1991). Deferoxamine is known to be an effective chelator for ferric iron.
MIDA can be prepared by converting MP 350 to the chloride by reaction with thionyl chloride (Bueckmann et al., Biotechnology & Applied Biochem., 9:258-268, 1987), and to the iminodiacetate by reaction of the chloride with sodium iminodiacetate (Wuenschell et al., J. Chromatog. 543: 345-354, 1991). The methyl ester can be prepared with methanolic HCl. However, alternative MP chelates, which are more effective chelators and are non-toxic, have been sought.
U.S. Pat. No. 6,020,373 discloses that the polyethylene glycol linked to a chelate methyl ester was a very effective protectant against radiation damage and doxorubicin toxicity in animal models. The well-known radioprotectant amifostine, S-2-[3-aminopropylamino]ethylphosphorothioate (WR-2721, Ethyol), that possesses a potential thiol, has been used extensively in the clinic for protection of normal tissues in radiotherapy and chemotherapy (Wasserman, T. H., Seminars in Oncology 21 (5 Supp. 11): 21-25, 1994), but its effectiveness is limited. Sulfhydryl compounds, such as cysteine and cysteamine, have been known to provide radiation protection in animals. Thiol groups scavenge radiation-produced free radicals by donating a hydrogen atom to damaged molecules. Despite extensive efforts to develop more effective protective agents, no thiol-based radioprotector has been found to be significantly better than cysteamine. Moreover, the use of thiol drugs to protect against radiation damage has been limited due to the toxicity of such compounds. Further, the use of esters of polar drugs to facilitate penetration into cells has been reported (Vos et al, Int. J. Radiat. Biol. Relat. Stud. 53:273-81, 1988).
A need clearly exists, therefore, for improved compositions and methods capable of protecting cells, tissues and organs against the damaging effects of an ionizing agent associated with radiation or chemotherapy, or with disease or other states in which the production of free radical oxidants such as peroxides, superoxide anions, hydroxyl radical or nitric oxide, or heavy metal cations is implicated.